The Raf protein serine/threonine kinase is a critical mediator of mitogenic signal transduction, and its altered expression or activity has been associated with several types of human cancer, including lung. We developed a mouse model for induction of this tumor type and examined the resultant tumors for Raf involvement. Tumors were induced by transplacental injection of 1-ethyl-1-nitrosourea followed by promotion with butylated hydroxytoluene. This regimen induces lung adenocarcinomas in 100% of offspring and T-cell lymphomas in 70%. Examination of tumors for altered expression or structure of a panel of oncogenes revealed consistent mutations of c-raf-1, along with a conspicuous lack of Ras mutations. Functional assays for mutated Raf-1 demonstrated that the mutations were activating. This is the first example of point mutated c- raf-1 in vivo, demonstrating a new class of activating raf mutations. Approximately 80% of tumors sequenced showed the same mutation, and we developed a rapid screen for identifying this alteration. The location of the mutations in subdomain XIII of the kinase domain, apparently at the surface of the substrate pocket, suggests that they might be exploited for the development of mutant-specific inhibitors. We have demonstrated the feasibility of targeting Raf in order to delay the onset of tumorigenesis by vaccination of mice with purified Raf protein. Initial examinations of human tumors also indicate the presence of Raf mutations.